Molecular Biology

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    Protocols in Current Issue
    A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s)
    Authors:  Antonia Efstathiou and Despina Smirlis, date: 01/20/2020, view: 77, Q&A: 0
    The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay ...
    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 87, Q&A: 0
    The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of ...
    Ribonucleoprotein Immunoprecipitation (RIP) Analysis
    Authors:  Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 197, Q&A: 0
    RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an ...
    A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s)
    Authors:  Antonia Efstathiou and Despina Smirlis, date: 01/20/2020, view: 77, Q&A: 0
    [Abstract] The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s ...
    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 87, Q&A: 0
    [Abstract] The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...
    Ribonucleoprotein Immunoprecipitation (RIP) Analysis
    Authors:  Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 197, Q&A: 0
    [Abstract] RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an RBP of interest is by immunoprecipitation (IP) analysis of native RNPs. RIP (RNP ...
    RNA Interactome Identification via RNA-BioID in Mouse Embryonic Fibroblasts
    Authors:  Joyita Mukherjee, Mirita Franz-Wachtel, Boris Maček and Ralf-Peter Jansen, date: 01/05/2020, view: 216, Q&A: 0
    [Abstract] Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA’s 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such ...
    Laser Capture Micro-dissection (LCM) of Neonatal Mouse Forebrain for RNA Isolation
    Authors:  Achira Roy, Mei Deng, Kimberly A. Aldinger, Ian A. GLass and Kathleen J. Millen, date: 01/05/2020, view: 278, Q&A: 0
    [Abstract] Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions in vivo. Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to ...
    Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
    Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 494, Q&A: 0
    [Abstract] Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...
    High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach
    Authors:  Franziska M. Heydenreich, Tamara Miljuš, Dalibor Milić and Dmitry B. Veprintsev, date: 01/05/2020, view: 270, Q&A: 0
    [Abstract] Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries ...
    A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae
    [Abstract] We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using ...
    Protocol for Ribosome Profiling in Bacteria
    Authors:  Fuad Mohammad and Allen R. Buskirk, date: 12/20/2019, view: 618, Q&A: 0
    [Abstract] Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome ...
    Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol
    Authors:  Alexandra R Crowe and Wei Yue, date: 12/20/2019, view: 720, Q&A: 0
    [Abstract] Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed ...



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